This invention is directed to a modified Factor VIII cDNA and its use for the improvement of the Factor VIII production.
Factor VII (FVIII) is a plasma coagulation cofactor implicated in the activation of Factor X (FX). A decrease in the presence or activity of Factor VIII in blood stream leads to hemophilia A. The level of the decrease in Factor VIII activity is directly proportional to the severity of the disease (Foster and T. S., 1989; Kaufman, 1992; Vlot et al., 1998). The current treatment of hemophilia A consists of the replacement of the missing protein by plasma-derived or recombinant FVIII. Recombinant FVIII is produced in CHO or BHK cells after selection of the best producing clones and amplification of the FVIII cDNA copy number.
Several studies have stressed the low FVIII production level in different cellular systems: Biosynthesis of FVIII was shown to be regulated in at least three different levels. First, among the FVIII cDNA sequence two nucleotides stretches, localized in the A2 coding domain, were demonstrated to act as transcriptional silencers (Fallaux et al, 1996; Hoeben et al., 1995; Koeberl et al., 1995; Lynch et al., 1993). Second, FVIII protein synthesis is tightly regulated by several reticulum endoplasmic chaperones (BiP; Calreticulin; Calnexin; ERGIC-53). Many of these interactions retain FVIII in the cell and direct it through the cellular degradation machinery (Dorner et al., 1987; Nichols et al., 1998; Pipe et al., 1998). Third, once secreted FVIII is sensitive to protease degradation and needs to be protected by von Willebrand Factor (vWF) (Kaufman et al., 1989).
It is therefore a problem to develop improved processes which result in higher yields of FVIII. The present invention offers a solution to this problem by a modified FVIII cDNA.